The field of the present invention is retroviruses and nucleic acid-free particles, methods of producing immunogenic retrovirus-like particles using baculovirus vectors, immunogenic compositions comprising retrovirus-like particles and methods for eliciting an immune response using compositions comprising retrovirus-like particles.
Assembly of virus particles of the lentivirus genus of retroviruses takes place by a budding process at the cellular plasma membrane. Representative lentiviruses include Simian Immunodeficiency Virus (SIV) and Human Immunodeficiency Virus (HIV). Studies with several retroviruses have demonstrated that the Gag protein expressed in the absence of other viral components is self-sufficient for particle formation and budding at the cell surface (Wills and Craven, 1991). Immature virus particles undergo a process of maturation wherein the viral protease cleaves the Gag precursor into the structural proteins: matrix, core and nucleocapsid proteins (Wills and Craven, 1991). It has been reported that the N-terminal region of the Gag precursor is a targeting signal for transport to the cell surface and membrane binding, which is required for virus assembly (Yuan et al., 1993; Zhou et al., 1994). The mechanism of specific incorporation of envelope protein in the virus particles is not understood, but interaction of Env with matrix protein p17 seems to be important (Yu et al., 1992; Dorfman et al., 1994).
Assembly of recombinant HIV-like particles that contain Gag structural proteins as well as Env glycoproteins gp120 and gp41 was previously reported using a vaccinia virus expression system (Haffar et al., 1990; Vzorov et al., 1991). These particles reportedly induce HIV-specific humoral and cellular immunity in rabbits (Haffar et al., 1991) and can inhibit virus production in latently infected peripheral blood mononuclear cells from HIV-1 seropositive donors (Haffar et al., 1992). Formation of retrovirus-like immature particles upon expression of the Gag precursor in insect cells using baculovirus vectors was demonstrated by several groups (Delchambre et al., 1989; Luo et al., 1990; Royer et al., 1991; Morikawa et al., 1991). These Gag particles resemble immature lentivirus particles which are efficiently assembled and released by budding from the insect cell membrane. In contrast to the expression in mammalian cells, inclusion of the protease region in Gag expressing vectors in the baculovirus system leads to overexpression of the protease and early processing of the Gag precursor into mature structural proteins within insect cells, which prevents particle formation (Morikawa et al., 1991; Hughes et al., 1993).
The expression patterns of retroviral envelope glycoproteins in the baculovirus system have several disadvantages in comparison to expression in mammalian cells. The proteins are cleaved very inefficiently and are mainly cell associated (Rusche et al., 1987; Wells and Compans, 1990; Hu et al., 1987). However, HIV-1 Env proteins produced in insect cells are immunologically and biologically active, as demonstrated by their ability to react specifically with immune serum (Hu et al., 1987) and to induce syncytium formation upon co-cultivation with HeLa T4 cells (Wells and Compans, 1990). Because of conditions used for purification of Env glycoproteins from baculovirus-infected insect cells, the preparations obtained often contain denatured gp160, which has little immunogenic activity (Moore et al., 1993). Nevertheless in some cases, as reported by Rusche et al. (1987), immunization with a crude lysate of HIV-1 Env expressing insect cells produced high titers of virus neutralizing and fusion blocking antibody in experimental animals.
The assembly of envelope proteins into HIV, SIV or other retrovirus-like particles has not been reported previously using baculovirus expression systems, possibly because of limitations in the transport and surface expression of the envelope glycoproteins.